Cryptococcus neoformans var. grubii: The In Vitro Antifungal Susceptibility Pattern in Addition to the Quantification of Phospholipase and Proteinase Enzymatic Activities

Abstract

Background: Cryptococcal meningoencephalitis is a life-threatening fungal infection in human immunodeficiency virus (HIV)-infected patients. Cryptococcus neoformans var. grubii and neoformans are the causative agents that usually respond well to fluconazole and amphotericin B. However, resistance/ non-responding cryptococcal meningitis cases to fluconazole and amphotericin B have been reported globally. Methods: The causative Cryptococcus was identified by phenotypic and singleplex polymerase chain reaction (PCR) targeting the putative sugar transporter (STR1) gene. In addition, the phospholipase and proteinase enzymatic activities of the isolates were determined by the plate method using egg yolk agar and bovine serum albumin agar plates, respectively. Finally, the in-vitro minimal inhibitory concentration (MIC) of fluconazole, voriconazole, and amphotericin B against isolated C. neoformans strains was determined by the broth microdilution method. Results: A total of 50 C. neoformans strains were isolated from the cerebrospinal fluid of HIV-infected patients, which were further identified as variety grubii by simplex polymerase chain reaction (PCR). All the isolated strains producing phospholipase and proteinase enzymes were determined by the calculation of Pz, a ratio of colony diameter and diameter of colony plus the precipitation zone. A comparative high proteinase enzyme activity was observed, and these strains produced medium to high phospholipase (mean Pz 0.3720±0.082, range 0.23-0.56) and proteinase activity (Mean Pz 0.3069±0.086, range 0.012- 0.54). A varied antifungal MIC was detected, and voriconazole had the lowest MIC50 and MIC90 (0.03 & 0.06 µg/mL) in comparison to fluconazole and amphotericin B. Conclusion: Cryptococcus neoformans var. grubii is the commonest cause of cryptococcal meningoencephalitis in HIV-infected patients. The isolates had varied extracellular hydrolytic enzyme activities. The emergence of C. neoformans strains with higher fluconazole MIC (≥4 mcg/mL) could have resulted in treatment failure.